Type I recessive congenital methaemoglobinaemia (RCM), caused by the reduced form of nicotinamide adenine dinucleotide (NADH)-cytochrome b(5) reductase (cytb(5)r) deficiency, manifests clinically as cyanosis without neurological dysfunction. Two mutations, E255- and G291D, have been identified in the NADH-binding lobe of cytb(5)r in previously reported patients, and we have detected a further novel mutation, D239G, in this lobe in two unrelated Irish families. Although one family belongs to the genetically isolated Traveller Community, which separated from the general Irish population during the 1845-48 famine, the D239G mutation was present on the same haplotype in both families. Three known cytb(5)r mutations were also identified, including the R159- mutation, which causes loss of the entire NADH-binding lobe and had previously been reported in an individual with type II RCM. Characterization of the three NADH-binding lobe mutants using a heterologous expression system revealed that all three variants retained stoichiometric levels of flavin adenine dinucleotide with spectroscopic and thermodynamic properties comparable with those of native cytb(5)r. In contrast to the E255- and G291D variants, the novel D239G mutation had no adverse impact on protein thermostability. The D239G mutation perturbed substrate binding, causing both decreased specificity for NADH and increased specificity for NADPH. Thus cytb(5)r deficient patients who are heterozygous for an NADH-binding lobe mutation can exhibit the clinically less severe type I phenotype, even in association with heterozygous deletion of the NADH-binding lobe.

Normal erythropoiesis is precisely regulated by a negative feedback mechanism based on the oxygen status of the body. Whenever a deficit in oxygen supply is detected erythropoietin (Epo) is rapidly synthesised to increase circulating red cells. This process is under the control of the HIF transcription complex, which is composed of one alpha and one beta subunit. Although the alpha subunit is constitutively synthesized, it is maintained at a low level by continual targeting to the proteasome. Prolines 402 and 564 in the oxygen dependent degradation (ODD) domain of HIF-1α are hydroxylated in the presence of oxygen by members of the PHD family of prolyl hydroxylases. Once hydroxylated HIF-1α is captured by the von Hippel Lindau tumour suppressor gene product (VHL), ubiquitylation follows and HIF-1α is then targeted for proteasomal degradation. Defects in the hypoxia response pathway appear to be the most common cause of erythrocytosis associated with inappropriately normal or elevated serum Epo.

The molecular basis of inherited erythrocytosis in most patients remains to be defined. Although all such patients have an absolute increase in red cell mass, their erythropoietin (EPO) levels differ widely, so they constitute a heterogeneous group of disorders known collectively as idiopathic erythrocytosis (IE). A proportion of individuals with IE progress to polycythemia vera (PV), a clonal disorder arising from a multipotent progenitor. Recently a gain-of-function mutation in Janus kinase 2 (JAK2), V617F, has been described in myeloproliferative disorders (MPD) and a stream of publications has confirmed its presence in the majority of patients with PV. Screening IE patients for this mutation will provide a useful additional means for delineating inherited and clonal disorders of erythrocytosis. Over the last decade we have maintained a registry of British and Irish erythrocytosis patients, consisting of clinical information and DNA samples obtained following full ethical approval. Screening the EPO receptor (EPO-R) in 120 patients identified one patient with a G6002A mutation, which leads to truncation of the receptor by 70 amino acids, increased sensitivity to EPO, and erythrocytosis. Screening the same patients for mutations in the von Hippel Lindau (VHL) gene has revealed individuals from 8 families of Asian origin who are homozygous for the Chuvash (R200W) mutation causing erythrocytosis. In addition, one Caucasian individual of English descent is compound heterozygous for R200W and the recently described G144R VHL mutation. A further individual, D1 (Percy et al, 2003 Blood 102:1097), of the same ethnicity is heterozygous for the Chuvash mutation and has been found to express the wild type allele. Both his mother and son, who are heterozygous for the Chuvash mutation, do not have IE, suggesting that the patient harbors a second unidentified genetic defect. Several such individuals have already been described (reviewed by Randi et al, 2005 Haematologica 90:689). In order to estimate the proportion of IE patients likely to progress to PV, 65 individuals from the registry with EPO levels in the low to normal range were screened by amplification refractory mutation system (ARMS) PCR for the MPD-associated V617F JAK2 mutation. Two individuals were positive, one of whom subsequently proceeded rapidly to PV, while the other has remained stable without any disease progression. In addition 9 families with VHL mutations were also screened and all were found to be negative for the JAK2 mutation, suggesting that the occurrence of these mutations tends to be mutually exclusive. Also the V617F JAK2 mutation does not constitute the second genetic defect in patient D1 who is heterozygous for the Chuvash VHL mutation. Although VHL mutations are the most frequent cause of inherited erythrocytosis in our registry they are present in only ~10% of patients, while the gain-of-function of JAK2 mutation is rare, leaving ~90% of the IE cases unexplained. Further study of this group may reveal additional regulatory mechanisms involved in red cell homeostasis.

The first congenital defect of hypoxia-sensing homozygosity for VHL 598C>T mutation was recently identified in Chuvash polycythemia. Subsequently, we found this mutation in 11 unrelated individuals of diverse ethnic backgrounds. To address the question of whether the VHL 598C>T substitution occurred in a single founder or resulted from recurrent mutational events in human evolution, we performed haplotype analysis of 8 polymorphic markers covering 340 kb spanning the VHL gene on 101 subjects bearing the VHL 598C>T mutation, including 72 homozygotes (61 Chuvash and 11 non-Chuvash) and 29 heterozygotes (11 Chuvash and 18 non-Chuvash), and 447 healthy unrelated individuals from Chuvash and other ethnic groups. The differences in allele frequencies for each of the 8 markers between 447 healthy controls (598C) and 101 subjects bearing the 598T allele (P < 10(-7)) showed strong linkage disequilibrium. Haplotype analysis indicated a founder effect. We conclude that the VHL 598C>T mutation, the most common defect of congenital polycythemia yet found, was spread from a single founder 1,000 to 62,000 years ago.

The Chuvash form of polycythemia is an autosomal recessive disorder common to a large number of families in central Russia. Affected individuals have been reported to be homozygous for an Arg200Trp mutation in the von Hippel-Lindau (VHL) gene. We have screened 78 patients with erythrocytosis and found 8 of Bangladeshi and Pakistani origin to be homozygous for the Arg200Trp mutation and another of English descent to be heterozygous. Of these patients, 5 have elevated serum erythropoietin (Epo) levels, while the other 4 have Epo values in the normal range. The heterozygous patient does not fulfill the Chuvash criterion for homozygosity of the Arg200Trp mutation and consequently may harbor a further, as yet uncharacterized, mutation. This mutation has a wider geographic distribution than originally presumed and haplotype analysis suggests a common origin of the Arg200Trp mutation in the 4 families, but it still remains to be established if it has arisen independently of the Chuvash population.

Acute promyelocytic leukemia (APL) is associated with a reciprocal and balanced translocation involving the retinoic acid receptor-alpha (RARalpha). All-trans retinoic acid (ATRA) is used to treat APL and is a potent morphogen that regulates HOX gene expression in embryogenesis and organogenesis. HOX genes are also involved in hematopoiesis and leukemogenesis. Thirty-nine mammalian HOX genes have been identified and classified into 13 paralogous groups clustered on 4 chromosomes. They encode a complex network of transcription regulatory proteins whose precise targets remain poorly understood. The overall function of the network appears to be dictated by gene dosage. To investigate the mechanisms involved in HOX gene regulation in hematopoiesis and leukemogenesis by precise measurement of individual HOX genes, a small-array real-time HOX (SMART-HOX) quantitative polymerase chain reaction (PCR) platform was designed and validated. Application of SMART-HOX to 16 APL bone marrow samples revealed a global down-regulation of 26 HOX genes compared with normal controls. HOX gene expression was also altered during differentiation induced by ATRA in the PML-RARalpha(+) NB4 cell line. PML-RARalpha fusion proteins have been reported to act as part of a repressor complex during myeloid cell differentiation, and a model linking HOX gene expression to this PML-RARalpha repressor complex is now proposed.